Waterlogging tolerance of five soybean genotypes through different physiological and biochemical mechanisms

Journal Pre-proof Waterlogging tolerance of five soybean genotypes through different physiological and biochemical mechanisms ´ Garcia, Cristiane Jovelina da-Silva, Kassia Luiza Teixeira Natalia ˜ Victor Cocco, Darwin Pomagualli, Fabiane Kletke de Oliveira, Joao ´ Lemos da Silva, Ana Claudia Barneche de Oliveira, Luciano do Amarante

PII:

S0098-8472(20)30001-0

DOI:

https://doi.org/10.1016/j.envexpbot.2020.103975

Reference:

EEB 103975

To appear in:

Environmental and Experimental Botany

Received Date:

30 September 2019

Revised Date:

6 January 2020

Accepted Date:

7 January 2020

Please cite this article as: Garcia N, da-Silva CJ, Teixeira Cocco KL, Pomagualli D, de Oliveira FK, da Silva JVL, de Oliveira ACB, do Amarante L, Waterlogging tolerance of five soybean genotypes through different physiological and biochemical mechanisms, Environmental and Experimental Botany (2020), doi: https://doi.org/10.1016/j.envexpbot.2020.103975

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

1

Waterlogging tolerance of five soybean genotypes through different physiological and biochemical mechanisms

Running Title: Waterlogging impacts on soybean genotypes

Natália Garcia1,* [email protected], Cristiane Jovelina da-Silva1, Kassia Luiza Teixeira Cocco1, Darwin Pomagualli1, Fabiane Kletke de Oliveira1,

ro of

João Victor Lemos da Silva1, Ana Cláudia Barneche de Oliveira2, Luciano do Amarante1

Department of Botany, Institute of Biology, Federal University of Pelotas, Capão do

-p

1

Leão, RS, Brazil

Brazilian Agricultural Research Corporation, Embrapa Temperate Climate

re

2

*

na

Corresponding author

lP

Agricultural Research Center, Pelotas, RS, Brazil

Highlights

Soybean genotypes suppressed waterlogging stress with distinct mechanisms.



PELBR10-6000 activated the fermentative enzymes.



BMX Potência restored metabolism to control levels at the end of the recovery.



PELBR11-6028 and PELBR11-6042 activated the antioxidant defenses in

Jo

ur



leaves. 

TEC IRGA 6070 was the only genotype that did not delay flowering.

2

Abstract Waterlogging is a serious environmental threat that limits crop growth and yield in low-lying, rainfed areas in many regions across the globe. Here we investigated the effects of waterlogging and subsequent re-oxygenation on the physiology and biochemistry of three soybean [Glycine max (L.) Merrill] genotypes (PELBR106000, PELBR11-6028, and PELBR11-6042) and two cultivars (TEC IRGA 6070 and

ro of

BMX Potência). Plants were grown under greenhouse conditions until the V4 stage when they were subjected to waterlogging for seven days. The water was then

drained and plants were allowed to recover for another seven days. Overall, all

-p

genotypes suppressed waterlogging stress with distinct mechanisms. Waterlogged

PELBR10-6000 surpassed control plant levels of CO2 assimilation rate and readily

re

responded to the energy lack induced by hypoxia by activating the fermentative

lP

enzymes and alanine aminotransferase. Similar mechanisms were observed in BMX Potência, which restored metabolism to control levels at the end of the recovery. PELBR11-6028 and PELBR11-6042 activated the antioxidant defenses, and TEC

na

IRGA 6070 did not delay flowering.

Keywords: abiotic stress; antioxidant activity; fermentative metabolism; Glycine

Jo

ur

max; hypoxia; leaf gas exchange.

1 INTRODUCTION Soybean [Glycine max (L.) Merrill] plants comprise a major source of protein

and oil for humans, livestock and various industrial products, with a total grain production of approximately 341 million tons worldwide (United States Department of Agriculture, 2019). Waterlogging is a widespread phenomenon drastically

3

reducing the growth and production of soybean in many regions of the world (Van Nguyen et al., 2017), mostly due to the occurrence of flat topography (Collaku & Harrison, 2002), high water tables and poor drainage of clay-like soils (Jitsuyama, 2017). In Brazil, ca. 28 million hectares are flood-prone (alluvial and hydromorphic soils), in which 13 million hectares are located in the lowlands of the Rio Grande do Sul state (Empresa Brasileira de Pesquisa Agropecuária, 2018). These areas are frequently used for rice farming, with manipulated flooding regimes (paddy rice)

ro of

(Jackson & Colmer, 2005), and soybean has been introduced as crop rotation to enhance the efficiency of the rice cropping system by improving the soil conditions and breaking pests, diseases and weed cycles.

-p

The negative impacts of waterlogging on crops is mostly a consequence of the slow diffusion rates of gases and the relatively low solubility of O2 in water

re

(Bailey-Serres & Voesenek, 2008; Limami, Diab & Lothier, 2014). One of the most

lP

severe problems encountered by plants subjected to waterlogging is the energy deficit as a result of root respiration inhibition caused by a shortage of O2 (van Dongen & Licausi, 2015). As a consequence, the increased levels of pyruvate

na

accumulated due to glycolysis activate the fermentative enzymes (Borella et al., 2019). Two important pathways induced under anaerobic conditions are lactic and

ur

ethanolic fermentation. The first is a one-step reaction from pyruvate to lactate,

Jo

catalyzed by lactate dehydrogenase (LDH) using NADH. The latter is a two-step process regenerating NAD+, in which pyruvate is first decarboxylated to acetaldehyde by pyruvate decarboxylase (PDC), and acetaldehyde is then converted to ethanol by alcohol dehydrogenase (ADH) (Zabalza et al., 2009; Zhang, Lyu, Jia, He, & Qin, 2017). Therefore, waterlogging and, ultimately, anaerobic metabolism potentially result in acute growth inhibition and even death in most crops due to

4

energy limitation, accumulation of toxic products (such as lactate) and carbon loss (via ethanol loss from roots) (Tamang, Magliozzi, Maroof, & Fukao, 2014). Several species such as soybean (Sousa & Sodek, 2003; Rocha et al., 2010a; Borella et al., 2017), Lotus japonicas L. (Rocha et al., 2010b), and Arabidopsis thaliana L. (Miyashita, Dolferus, Ismond, & Good, 2007) accumulate alanine, an amino acid produced by the enzyme alanine aminotransferase (AlaAT), under hypoxia. The synthesis of alanine plays an important role in regulating the glycolytic

ro of

flux by preventing the excessive accumulation of pyruvate (Zabalza et al., 2009) while retaining carbon and nitrogen resources within the cell (Rocha et al., 2010b). Unlike the production of lactate and ethanol, alanine accumulation does not have

-p

detrimental side effects on the cell.

Under waterlogging conditions, plants also exhibit reduced stomatal

re

conductance (gs) (Posso, Borella, Reissing, & Baccarin, 2018; Barickman, Simpson,

lP

& Sams, 2019), which is often accompanied by reduced net CO2 assimilation and leaf chlorosis (de Souza, Souza, Dousseau, Castro, & Magalhães, 2013; Posso et al., 2018). Impaired net CO2 accumulation accompanied by the limited capacity to

na

absorb water and nutrients represent additional factors reducing plant growth and biomass accumulation (Marashi, 2018; Ploschuk, Miralles, Colmer, Ploschuk, &

ur

Striker, 2018; Ye et al., 2018).

Jo

In addition to the various negative effects of waterlogging, as water level

descends due to soil drainage, roots are suddenly exposed to elevated oxygen levels resulting in redox imbalance and overproduction of reactive oxygen species (ROS) in mitochondria and chloroplasts (Halliwell, 2006). To counteract the hazardous effects of ROS, plants have evolved a complex antioxidant system comprised by enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase

5

(CAT) (Fukao, Barrera-Figueroa, Juntawong, & Peña-Castro 2019), and nonenzymatic scavengers, such as carotenoids (Doupis, Kavroulakis, Psarras, & Papadakis, 2017). Previous studies using wheat (Triticum aestivum L.) (Malik, Colmer, Lambers, Setter, & Schortemeyer, 2002), barley (Hordeum vulgare L.) (de San Celedonio, Abeledo, & Miralles, 2014), and rapeseed (Brassica napus L.) (Wollmer, Pitann, & Mühling, 2017) observed that plants exposed to waterlogging at the

ro of

vegetative phase exhibit reduced growth rate which demonstrates their sensitivity in early vegetative stages (Xu et al., 2015). Given that few studies addressed the effects of waterlogging on soybean plants at the vegetative stage (Mutava et al., 2015;

-p

Andrade, Souza, Santos, Silva, & Alves, 2018), we aimed to describe the influence of waterlogging on the physiology and metabolism of leaves and roots of five

lP

re

soybean genotypes during and after waterlogging stress.

2 MATERIAL AND METHODS

2.1 Plant material and growth conditions

na

To perform this study, we selected two commercial cultivars, BMX Potência (POT) and TEC IRGA 6070 (6070), often cultivated in lowland areas in Southern

ur

Brazil, and three lineages from Embrapa Soybean Breeding Program PELBR10-6000

Jo

(6000), PELBR11-6028 (6028) and PELBR11-6042 (6042). The experiment was carried out in a greenhouse at Embrapa Lowlands Experimental Station (see environmental conditions in Figure S1 and Table S1), Capão do Leão (31º80'36"S and 52º41'12"W), Brazil, from December 2016 to January 2017. Seeds were inoculated on sowing with Bradyrhizobium japonicum, strain SEMIA 5079 (BIOAGRO). Plants were cultivated from seeds in 3 L pots (two plants

6

per pot) containing vermiculite and watered daily. Twice a week, they received 200 mL of N-free nutrient solution at full strength (Hoagland & Arnon, 1950). Plants were subjected to waterlogging 30 d after sowing at the V4 stage (Fehr, Caviness, Burmood, & Pennington, 1971). Waterlogging was imposed during seven days by placing each pot (with drainage holes) into non-punctured pots of the same dimension and filling the pots with 1/3 full strength N-free nutrient solution until the water level reached 2 cm above the vermiculite surface. The nutrient solution was

ro of

monitored daily and refilled when necessary. The O2 concentration in solution was monitored with an O2 meter (Handylab OX1, Hofheim, Germany). After seven days of waterlogging, the pots were drained and plants were allowed to recover for

-p

additional seven days. Control plants were not subjected to waterlogging.

Plants were sampled during waterlogging at days two and seven, and during

re

the recovery at days two and seven. During harvesting, roots were carefully washed

lP

and homogeneous healthy leaves of the same size and age (first fully expanded trifoliate) were harvested, weighed and stored frozen (-80ºC) until biochemical analysis.

na

2.2 Plant growth, leaf gas exchange and water potential Stem diameter (SD) was assessed using a caliper rule, while root dry matter

ur

(RDM) and shoot dry matter (SDM) were obtained placing samples in an oven at

Jo

65ºC for 72 h. Pre-dawn leaf water potential (WP) was assessed prior to sunrise using a pressure pump (SEC-3115-P40G4V, Soil moisture, Santa Barbara, USA). Net CO2 assimilation rate (A), stomatal conductance (gs) and transpiration rate

(E) were performed from 11:00 to 12:00 h, using a portable open-flow gas exchange system (LI-6400XT, LI-COR, Lincoln, USA). Conditions in the leaf cuvette were set

7

at relative humidity of ca. 50%, light intensity of 1000 µmol m-2 s-1, temperature of 25ºC and CO2 of 400 ppm. 2.3 Antioxidant enzymes Leaves and roots (0.250 g) were ground into powder using 100 mM potassium phosphate buffer (pH 7.8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ascorbic acid (AsA) and 10% (w/w) polyvinylpyrrolidone (PVPP). Then, the homogenate was centrifuged at 12.000 × g at 4ºC for 20 min and the

ro of

supernatant was used for enzyme activity measurements. SOD (EC 1.15.1.1) activity was estimated by the ability of the enzyme to

inhibit the nitroblue-tetrazolium (NBT) reduction (Giannopolitis & Ries, 1997) in a

-p

reaction medium consisting of enzyme extract, 86.5 mM potassium phosphate buffer (pH 7.8), 14 mM methionine, 0.1 M EDTA, 5.6 µM NBT and 0.2 µM riboflavin.

re

One unit of SOD activity was defined as the amount of enzyme that caused 50%

lP

inhibition of the reduction of NBT. CAT (EC 1.11.1.6) activity was assayed by measuring the initial rate of disappearance of hydrogen peroxide (H2O2) (Havir & McHale, 1987). The incubation mixture contained crude enzyme extract, 100 mM

na

potassium phosphate buffer (pH 7.0) and 12.5 mM H2O2. The measurements were recorded at 240 nm and the enzyme activity was calculated using the molar

ur

extinction coefficient (ε = 36 µM-1 cm-1). APX (EC 1.11.1.11) activity was measured

Jo

following the method of Nakano & Asada (1981). The assay mixture contained crude enzyme extract, 37.5 mM potassium phosphate buffer (pH 7.0), 0.25 mM AsA and 5 mM H2O2. The absorbance was recorded at 290 nm and the activity of APX was calculated using ε = 2.8 mM-1 cm-1. The protein content was determined according to Bradford (1976) using bovine serum albumin as the standard. 2.4 H2O2 content

8

Leaves and roots (0.250 g) were ground with 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged (12,000 × g; 4ºC; 20 min) and the supernatant was used for the analyses according to Velikova, Yordanov, and Edreva (2000). The samples were incubated in a reaction medium containing 10 mM potassium phosphate buffer (pH 7.0) and 1.0 M potassium iodide at 30ºC for 10 min. The absorbance was measured at 390 nm. A calibration curve was obtained with H2O2 standard.

ro of

2.5 Fermentative enzymes and AlaAT Frozen roots (0.250 g) were ground into powder using an extraction buffer (50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol and 3% (w/w) PVPP). The

-p

homogenate was centrifuged at 12,000 × g at 4ºC for 20 min, and an aliquot of the

supernatant was desalted using a PD-10 column (GE Healthcare, Buckinghamshire,

re

UK). The eluted protein fraction was used to determine ADH (EC 1.1.1.1), PDC (EC

lP

4.1.1.17), LDH (EC 1.1.1.17) and AlaAT (EC 2.6.1.2) activities. The enzymatic assays were performed by monitoring the oxidation of NADH at 340 nm. ADH and PDC activities were assayed following the method proposed by

na

Hanson, Jacobsen, and Zwar (1984) with some modifications. For ADH, the reaction mix contained 50 mM phosphate buffer (pH 7.0), 0.6 mM NADH and 5.0 mM

ur

acetaldehyde. The PDC assay was monitored in a reaction mix containing 50 mM

Jo

MES buffer (pH 6.0), 0.2 mM NADH, 0.5 mM thiamine pyrophosphate, 1 mM magnesium chloride, 20 mM oxamic acid, 5 units of ADH and 10 mM Na-pyruvate. The LDH assay followed the method proposed by Hanson and Jacobsen (1984) with some modifications: the reaction was monitored in a reaction mix containing 50 mM buffer Tris-HCl (pH 7.5), 0.6 mM NADH, 3.0 µM potassium KCN, 0.2 mM 4methylpyrazole and 10 mM Na-pyruvate. The AlaAT assay contained 10 mM L-

9

alanine, 5 mM 2-oxoglutarate, 0.6 mM NADH, 50 mM Tris/HCl (pH 7.5) and 5 units of lactate dehydrogenase, as described by Good and Muench (1992). 2.6 Photosynthetic pigments Chlorophyll a (chl a), chlorophyll b (chl b) and carotenoids were extracted from leaves using dimethyl sulfoxide as described by Wellburn (1994). 2.7 Experimental design and statistics Pots were arranged in a complete randomized design with five replicates. The

ro of

two plants in each pot were pooled together to form a single replicate. For each treatment group (2W, 7W, 2R, and 7R), there was a control group of plants (2WC, 7WC, 2RC, and 7RC). Each treatment was compared to its corresponding control

-p

(e.g. 2W vs 2WC).

The data were analyzed by two-way ANOVA and the means were compared

re

by Student’s t-test (P ≤ 0.05). Statistical analyses were performed using SigmaPlot

lP

11.0 (Systat Software Inc., San Jose, California). Results are shown as treatment/control (T/C) ratio resulting in the waterlogging tolerance index proposed by Jitsuyama (2017). This ratio was transformed by log10 according to Goufo et al.

na

(2017). T/C ratios greater than zero indicate that the means were higher in the treatment than in the control group, while T/C ratios lower than zero showed that

ur

control plants presented higher values than those treated (waterlogging or recovery).

Jo

For water potentials, which present negative values, the contrary is applied. Ratios close to zero indicate similar values for control and treatment. The absolute value indicates the magnitude of the difference.

3. RESULTS

10

During the first two days of waterlogging, O2 levels decreased markedly in the rooting medium, while from day two to seven, decreases in the O2 level were much less sharp (Figure S2). Waterlogging limited root biomass accumulation in a genotype-dependent manner (Figure 1A; Table S2). Genotype 6028 presented the greatest difference in root biomass under waterlogging compared to control, while genotype 6000 presented the least difference. Draining allowed an increase in the accumulation of

ro of

root biomass, resulting in a lower difference in root biomass between recovered and control plants. After seven days of recovery, genotypes 6070 and POT exhibited

similar root dry mass to control plants. Waterlogging and recovery did not affect

-p

shoot biomass production of all genotypes in a similar fashion (Figure 1B). While POT plants increased shoot biomass during waterlogging, the other genotypes

re

presented similar shoot biomass to control plants. During recovery, genotype 6000

lP

increased and genotypes 6070 and POT decreased shoot biomass production compared to control. The stem diameter of all genotypes consistently increased during waterlogging (Figure 1C). Seven days of recovery resulted in a similar stem

na

diameter between control and recovery plants due to decreases in the stem diameter of waterlogged plants (Table S2). Most genotypes subjected to waterlogging

ur

followed by recovery, except for 6070, exhibited a delayed development in terms of

Jo

flowering at the end of the experiment (Table S3). Overall, waterlogging significantly decreased the leaf gas exchange of

soybean plants (Figure 2, Table S4). The A was lower for almost all genotypes when plants were subjected to waterlogging for two days when compared to control, and consistently lower for all genotypes subjected to waterlogging for seven days (Figure 2A). Plants subjected to waterlogging presented A lower than control at two days of

11

recovery, but similar or even higher A (genotype 6000) at seven days of recovery. Two days of waterlogging resulted in stomatal closure only for the genotype 6028, consistent with the largest reduction in E (Figure 2B-C). Waterlogging for seven days resulted in stomatal closure and decreased E for most genotypes (genotypes 6028, 6042, 6070). On day seven of recovery, all genotypes presented similar or higher gs and E than control. Regarding WP, only genotype 6028 presented WP different (higher) to control at day two of waterlogging (Figure 2D). On day seven of

ro of

waterlogging, genotype 6028 was maintained at higher WP than control. On day two of recovery, all genotypes exhibited higher WP than control, and on day seven genotypes 6000, 6028 and 6070 remained more hydrated than control.

-p

Waterlogging resulted in lower amounts of photosynthetic pigments in a

genotype-dependent manner (Figure 3, Table S5). The Chl a level decreased with

re

waterlogging and seven days of recovery allowed plants to restore it to control levels

lP

(Figure 3A). Plants of genotypes 6000, 6070 and POT subjected to waterlogging also presented lower amounts of Chl b on day two of recovery, with plants of genotype 6000 also presenting lower levels of this molecule on day seven of recovery (Figure

na

3B). Carotenoid levels were not reduced by two days of waterlogging (Figure 3C). Seven days of waterlogging, on the other hand, resulted in decreased carotenoid

ur

levels for genotypes 6000, 6028 and 6042. Seven days of recovery resulted in similar

Jo

or even higher (POT) levels of carotenoids for all genotypes. The activity of SOD and CAT in leaves were the most affected antioxidant

enzymes during waterlogging and recovery (Figure 4, Table S6). The activity of SOD increased in leaves at seven days of waterlogging for all genotypes, but POT (Figure 4A). All genotypes presented similar or higher (genotypes 6028 and 6042) SOD activity at seven days of recovery. Waterlogging and recovery did not affect the

12

activity of APX in a consistent and significant fashion for leaves (Figure 4B). The CAT activity in leaves was modified in a genotype- and condition-manner (Figure 4C). Genotype 6000 increased CAT activity when compared to control at two days of recovery and decreased at seven days of recovery. Genotype 6028 decreased CAT activity within seven days of waterlogging and increased on day two of recovery. Genotype 6042 increased CAT activity within two days of waterlogging and at seven days of recovery. The CAT activity was

ro of

slightly reduced at two days of waterlogging in leaves of the genotype 6070 and remained

similar to control during the rest of the experiment. POT plants reduced CAT activity

-p

at seven days of waterlogging and recovery. Waterlogging and recovery did not significantly affect the activity of SOD and APX in roots (Figures 4D-E). The

re

activity of CAT in roots increased at the beginning of waterlogging (2 d) for

lP

genotypes 6000, 6028 and POT, and remained unaltered and lower during the rest of the experiment (Figure 4F).

The H2O2 content in leaves did not change within two days of waterlogging

na

and increased at seven days of waterlogging and two of recovery for all genotypes (Figure 5A and Table S7). Only genotype 6042 maintained high levels of H2O2 at the

ur

end of the experiment. The H2O2 content in roots increased only on day two of

Jo

waterlogging for genotype 6000 and day two of recovery for genotype 6070 (Figure 5B). The remaining genotypes exhibited similar or lower levels of H2O2 over the course of the experiment. Increases in LDH activity were observed at two days of waterlogging for genotypes 6000, 6028 and 6042, at two days of recovery for POT, and at seven days of recovery for 6070 (Figure 6A, Table S8). The activity of ADH and PDC increased

13

at seven days of waterlogging (Figure 6B-C), except for genotype 6028, which increased PDC, but not ADH. At the end of the recovery, the activity of these enzymes was similar or lower than the control for almost all genotypes. The AlaAT activity increased only at day seven of waterlogging and two of recovery for 6000 and day two of waterlogging for POT (Figure 6D). 4. DISCUSSION Waterlogging for seven days did not result in plant mortality or in severe

ro of

limitation in plant biomass accumulation for any genotype. Overall, all genotypes exhibited moderate tolerance to waterlogging through distinct mechanisms to

overcome the waterlogging-induced hypoxia stress. While some genotypes used

-p

several tolerance mechanisms during waterlogging and recovery (e.g. 6000 and

mechanisms (e.g. 6070 and POT).

re

6028), others overcame the stress by exhibiting less alteration regarding such

lP

Waterlogging negatively affected root biomass accumulation of almost all genotypes. Regarding shoot biomass, most genotypes presented shoot biomass similar to the control, and POT waterlogged plants accumulated more shoot biomass.

na

The investment in shoot growth is an interesting mechanism of waterlogging tolerance, possibly related to metabolites sent to the shoot via the xylem, permitting

ur

the removal of electrons from the hypoxic root (Vitor & Sodek, 2019).

Jo

Lower water uptake by the roots due to lower gs is one of the main factors

limiting plant growth (Barickman et al., 2019). The higher WP for POT plants during waterlogging may have contributed to maintaining gs and E similar to the control, as well as may have promoted higher shoot growth. In the meantime, the lower WP found in 6028 plants likely resulted in stomatal closure and proportionally higher inhibition of root biomass accumulation. Ren, Zhang, Dong, Liu, and Zhao (2016)

14

suggest that reductions in photosynthesis after waterlogging are mainly due to stomatal factors, which can be confirmed by our data. Most genotypes presented decreases in A accompanied by stomatal closure during waterlogging, however, decreases in A in POT waterlogged plants were not driven by stomatal limitations, since gs did not decrease upon waterlogging. For this genotype, decreases in A were probably due to decreases in chl a content. Indeed, in this genotype visible yellowing of leaves was noticed during waterlogging indicating chlorophyll degradation.

ro of

Reductions in chlorophyll content may be related to nitrogen deficiency. According to Amarante, Lima and Sodek (2006), nitrogen fixation in soybean plants under waterlogging is inhibited almost immediately due to the decrease in O2 supply.

-p

Limited nitrogen content in plants results in the remobilization of nitrogen from older to younger leaves. Decreases in chl a were also observed to the genotypes 6000,

re

6028 and 6042. In addition, genotypes 6000, 6028 and 6042 also decrease carotenoid

lP

content. Genotype 6070 presented normal levels of chl a and chl b and carotenoids under waterlogging, as well as genotype POT for carotenoid content. On the other hand, genotype 6028 presented high levels of carotenoids. Carotenoids probably

na

acted as an antioxidant defense protecting photosynthetic apparatus from ROS (Li et al., 2012) and may have been an important antioxidant defense for genotype 6028.

ur

The lower content of photosynthetic pigments, as well as the decreases in A,

Jo

can be caused by ROS accumulation. Indeed, the content of H2O2 increased in leaves of all genotypes under waterlogging. The H2O2 accumulation can be related to the increase in SOD activity, which in turn may have activated CAT (for genotype 6042) and APX (for genotype 6028). In roots, the content of H2O2 increased during waterlogging just for genotype 6000, however, the H2O2 level was not enough to activate scavenger enzymes. Regarding re-oxygenation, soybean plants did not

15

present ROS accumulation and oxidative damage in roots. On the other hand, the levels of H2O2 in leaves increased in all genotypes. Similar to leaves of waterlogged plants, the overproduction of ROS in leaves of plants under re-oxygenation may be a result of impared photosynthesis, resulting from reduced stomatal opening and pigment damage. Light-harvesting under these conditions leads to an overload of the electron transport chain in chloroplasts, leading to electron leakage and ROS accumulation (Gill & Tuteja, 2010).

ro of

At the end of the recovery, only genotype 6042 still had high levels of H2O2. In all other genotypes, H2O2 levels were restored due to the high activity of the

enzymes APX (for genotype 6070) and CAT (for genotypes 6000 and 6028) and the

-p

high levels of carotenoids (for genotype POT), an important non-enzymatic

antioxidant (Young & Lowe, 2018). Oxidative control was efficient in leaves, given

re

that at the end of recovery, chl a and b and carotenoid levels were restored (except

lP

genotype 6000 which still had low chl b levels). Consequently, A, gs, E and WP were also restored to control levels.

During hypoxia, fermentative enzymes prevent pyruvate accumulation,

na

contribute to NADH cycling, and produce substrate-level ATP (Borella, Durigon, Amarante, & Emygdio, 2013; Bui et al., 2019). However, the first pathway enzyme,

ur

LDH, produces lactate, which acidifies the cytosol of cells (Banti et al., 2013). The

Jo

genotypes 6000, 6028 and 6042 increased LDH under waterlogging. Lactate induces PDC activity, which converts pyruvate in acetaldehyde, resulting in ethanol production by the subsequent activity of ADH. Both enzymes were activated in genotypes 6000, 6042, 6070 and POT. Komatsu et al. (2009) detected an upregulation of ADH and PDC genes in soybean under waterlogging, suggesting that these genes are involved in the perception of O2 deprivation and exhibit a similar

16

enzyme cascade as other species under hypoxia. PDC activity in genotype 6028 increased after two and seven days of waterlogging. However, increases in ADH activity was not observed during waterlogging. AlaAT increased only in genotypes 6000 and POT, likely as a tolerance mechanism since alanine metabolism can prevent pyruvate accumulation, facilitating the continued operation of glycolysis during waterlogging (Rocha et al., 2010b; Borella et al., 2017). Regarding the activity of the fermentative enzymes in the recovery period,

ro of

genotypes 6028 and 6070 remained with higher LDH activity than control. In addition, genotypes 6070 and 6000 still maintained high activities of PDC and AlaAT, respectively. ADH activity was completely restored at the end of the

-p

recovery period. At the beginning of the recovery period and the end of the

experiment, LDH, PDC, and AlaAT activities were maintained guaranteeing ATP

re

production and preventing future hypoxic stress (Zhang et al., 2017).

lP

Facilitating the O2 entry into the roots is an important way to prevent larger cell damage. Soybean plants exhibited thickened stems presumably because of the aerenchymatous tissue induced by waterlogging as reported by Thomas, Guerreiro,

na

and Sodek (2005), and Shimamura, Yamamoto, Nakamura, Shimada, and Komatsu (2010). In nodulated legumes, a large number of studies have shown the beneficial

ur

effects of aerenchyma towards nitrogen fixation under waterlogging conditions

Jo

(Minchin & Summerfield, 1976; James & Sprent, 1999; Shimamura, Mochizuki, Nada, & Fukuyama, 2002), by providing a pathway for the diffusion of gases to the submerged nodules. At the end of the experiment, all genotypes were able to overcome waterlogging stress, presenting most of the parameters analyzed at control levels. However, only genotype 6070 showed no developmental delay in terms of flowering

17

at the end of the stress period. The 6000 genotype was the one that delayed flowering finishing the evaluation in V10. The other genotypes were in transition stages (V10/R1). These data reveal the damaging effect of waterlogging on soybean plant growth and development and demonstrate the importance of evaluating the developmental stage of plants upon waterlogging stress, as developmental delays

ro of

may compromise grain harvesting (Ploschuk et al., 2018).

AUTHOR STATEMENT

N.G., K.L.T.C., and J.V.L.S. performed the greenhouse experiment, as well as the

-p

samplings and analyses of growth. N.G., K.L.T.C., D.P., and F.K.O. performed biochemistry analyses. L.A. and A.C.B.O. supervised the project. L.A. and C.J.S.

na

Declaration of interests

lP

manuscript with help from L.A.

re

provided critical feedback and helped shape the research. N.G. and C.J.S. wrote the

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Jo

ur

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

ACKNOWLEDGMENTS

18

We are grateful to the Brazilian Agricultural Research Corporation (EMBRAPA) for soybean genotype seeds. This study was financed in part by Coordination for the

Jo

ur

na

lP

re

-p

ro of

Improvement of Higher Level Personnel (CAPES) – “Finance Code 001".

19

REFERENCES Amarante, L. do, Lima, J. D., & Sodek, L. (2006). Growth and stress conditions cause similar changes in xylem amino acids for different legume species. Environmental and Experimental Botany, 58, 123–129. doi: 10.1016/j.envexpbot.2005.07.002 Andrade, C. A., Souza, K. R. D. de, Santos, M. de O., Silva, D. M. da, & Alves, J. D. (2018). Hydrogen peroxide promotes the tolerance of soybeans to waterlogging.

ro of

Scientia Horticulturae, 232, 40–45. doi: 10.1016/j.scienta.2017.12.048 Bailey-Serres, J., & Voesenek, L. A. C. J. (2008). Flooding Stress: Acclimations and Genetic Diversity. Annual Review of Plant Biology, 59, 313–339. doi:

-p

10.1146/annurev.arplant.59.032607.092752

Banti, V., Giuntoli, B., Gonzali, S., Loreti, E., Magneschi, L., Novi, G., ... Perata, P.

re

(2013). Low oxygen response mechanisms in green organisms. International

lP

journal of molecular sciences, 14, 4734–4761. doi: 10.3390/ijms14034734 Barickman, T. C., Simpson, C. R., & Sams, C. E. (2019). Waterlogging Causes Early Modification in the Physiological Performance, Carotenoids, Chlorophylls,

na

Proline, and Soluble Sugars of Cucumber Plants. Plants, 8, 160. doi: 10.3390/plants8060160

ur

Borella, J., Becker, R., Lima, M. C., de Oliveira, D. S. C., Braga, E. J. B., de

Jo

Oliveira, A. C. B., & do Amarante, L. (2019). Nitrogen source influences the antioxidative system of soybean plants under hypoxia and re-oxygenation. Scientia Agricola, 76, 51–62. doi: 10.1590/1678-992x-2017-0195

Borella, J., Durigon, M. A., Amarante, L. D., & Emygdio, B. M. (2013). Fermentative enzymes activity in soybean roots and nodules under hypoxia and

20

post-hypoxia conditions. Ciência Rural, 43, 970–977. doi: 10.1590/S010384782013000600005 Borella, J., Oliveira, H. C., de Oliveira, D.S.C., Braga, E.J.B., de Oliveira, A.C.B., Sodek, L., & do Amarante, L. (2017). Hypoxia-driven changes in glycolytic and tricarboxylic acid cycle metabolites of two nodulated soybean genotypes. Environmental and Experimental Botany, 133, 118–127. doi: 10.1016/j.envexpbot.2016.10.007

ro of

Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemical, 72, 248–254. doi: 10.1016/0003-2697(76)90527-3

-p

Bui, L. T., Novi, G., Lombardi, L., Iannuzzi, C., Rossi, J., Santaniello, A., ...

Zaffagnini, M. (2019). Conservation of ethanol fermentation and its regulation in

re

land plants. Journal of experimental botany, 70, 1815–1827. doi:

lP

10.1093/jxb/erz052

Collaku, A., & Harrison, S. A. (2002). Losses in wheat due to waterlogging. Crop Science, 42, 444–450. doi: 10.2135/cropsci2002.4440

na

de San Celedonio, R. P., Abeledo, G. L., & Miralles, D. J. (2014). Identifying the critical period for waterlogging on yield and its components in wheat and barley.

ur

Plant and Soil, 378, 265–277. doi: 10.1007/s11104-014-2028-6

Jo

de Souza, T. C., Souza, E. D., Dousseau, S., de Castro, E. M., & Magalhaes, P. C. (2013). Seedlings of Garcinia brasiliensis (Clusiaceae) subjected to root flooding: physiological, morphoanatomical, and antioxidant responses to the stress. Aquatic Botany, 111, 43–49. doi: 10.1016/j.aquabot.2013.08.006 Doupis, G., Kavroulakis, N., Psarras, G., & Papadakis, I. E. (2017). Growth, photosynthetic performance and antioxidative response of ‘Hass’ and ‘Fuerte’

21

avocado (Persea americana Mill.) plants grown under high soil moisture. Photosynthetica, 55, 655–663. doi: 10.1007/s11099-016-0679-7 Empresa Brasileira de Pesquisa Agropecuária. (n.d.). Projeto Soja Encharcamento. Retrieved from https://bioinfo.cnpso.embrapa.br/flooding/ Fehr, W. R., Caviness, C. E., Burmood, D. T., & Pennington, J. S. (1971). Stage of development descriptions for soybeans, Glycine max (L.) Merrill. Crop Science, 11, 929–931. doi: 10.2135/cropsci1971.0011183X001100060051x

ro of

Fukao, T., Barrera-Figueroa, B. E., Juntawong, P., & Peña-Castro, J. M. (2019). Submergence and Waterlogging Stress in Plants: A Review Highlighting

Research Opportunities and Understudied Aspects. Frontiers in Plant Science, 10,

-p

340. doi:10.3389/fpls.2019.00340

Giannopolitis, C. N., & Ries, S. K. (1977). Superoxide dismutases: I. Occurrence in

re

higher plants. Plant Physiology, 59, 309–314.

lP

Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48, 909–930. doi: 10.1016/j.plaphy.2010.08.016

na

Good, A. G., & Muench, D. G. (1992). Purification and characterization of an aerobically induced alanine aminotransferase from barley roots. Plant Physiology,

ur

99, 1520–1525. doi: 10.1104/pp.99.4.1520

Jo

Goufo, P., Moutinho-Pereira, J. M., Jorge, T. F., Correia, C. M., Oliveira, M. R., Rosa, E. A. S., ... Trindade, H. (2017). Cowpea (Vigna unguiculata L. Walp.) Metabolomics: Osmoprotection as a Physiological Strategy for Drought Stress Resistance and Improved Yield. Frontiers in Plant Science, 8, 586. doi: 10.3389/fpls.2017.00586

22

Halliwell, B. (2006). Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiology. 141, 312–322. doi:10.1104/pp.106.077073 Hanson, A. D., & Jacobsen, J.V. (1984). Control of lactate dehydrogenase, lactate glycolysis, and α-amilase by O2 defcit in barley aleurone layers. Plant Physiology, 74, 566–572. doi: 10.1104/pp.75.3.566 Hanson, A. D., Jacobsen, J. V., & Zwar, J. A. (1984). Regulated expression of three

ro of

alcohol dehydrogenase genes in barley aleurone layers. Plant Physiology, 75, 573–581. doi: 10.1104/pp.75.3.573

Havir, E. A., & McHale, N. A., (1987). Biochemical and developmental

-p

characterization of multiple forms of catalase in tobacco leaves. Plant Physiology, 84, 450–455.

re

Hoagland, D. R., & Arnon, D. I. (1950). The Water Culture Method of Growing

California.

lP

Plants without Soil. Berkeley, CA: College of Agriculture, University of

Jackson, M.B., & Colmer, T.D. (2005). Response and adaptation by plants to

na

flooding stress. Annals of Botany, 96, 501–505. doi: 10.1093/aob/mci205 James, E. K., & Sprent, J. I. (1999). Development of N2-fixing nodules on the

ur

wetland legume Lotus uliginosus exposed to conditions of flooding. New

Jo

Phytologist, 142, 219–231. doi: 10.1046/j.1469-8137.1999.00394.x

Jitsuyama, Y. (2017). Hypoxia-Responsive Root Hydraulic Conductivity Influences Soybean Cultivar-Specific Waterlogging Tolerance. American Journal of Plant Sciences, 8, 770–790. doi: 10.4236/ajps.2017.84054 Komatsu, S., Yamamoto, R., Nanjo, Y., Mikami, Y., Yonukawa, H., & Sakata, K. (2009). A comprehensive analysis of the soybean genes and proteins expressed

23

under flooding stress using transcriptome and proteome techniques. Journal of Proteome Research, 8, 4766–4778. doi: 10.1021/pr900460x Li, X., Zhang, L., Li, Y., Ma, L., Bu, N., & Ma, C. (2012). Changes in photosynthesis, antioxidant enzymes and lipid peroxidation in soybean seedlings exposed to UV-B radiation and/or Cd. Plant and Soil, 352, 377–387. doi: 10.1007%2Fs11104-011-1003-8 Limami, A. M., Diab, H., & Lothier, J. (2014). Nitrogen metabolism in plants under

ro of

low oxygen stress. Planta, 239, 531–541. doi: 10.1007/s00425-013-2015-9 Malik A. I., Colmer T. D., Lambers H., Setter T.L., & Schortemeyer M. (2002).

Short-term waterlogging has long-term effects on the growth and physiology of

-p

wheat. New Phytologist, 153, 225–236. doi: 10.1046/j.0028-646X.2001.00318.x Marashi, S. K. (2018). Evaluation of uptake rate and distribution of nutrient ions in

re

wheat (Triticum aestivum L.) under waterlogging condition. Iranian Journal of

lP

Plant Physiology, 8, 2539–2547. doi: 10.22034/IJPP.2018.543416 Minchin, F. R., & Summerfield, R. J. (1976). Symbiotic nitrogen fixation and vegetative growth of cowpea (Vigna unguiculata (L.) Walp.) in waterlogged

na

conditions. Plant and Soil, 45, 113–127. doi: 10.1007/BF00011133 Miyashita, Y., Dolferus, R., Ismond K. P., & Good A. G. (2007). Alanine

ur

aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis

Jo

thaliana. The Plant Journal, 49, 1108–1121. doi: 10.1111/j.1365313X.2006.03023.x

Mutava, R. N., Prince, S. J. K., Syed, N. H., Song, L., Valliyodan, B., Chen, W., & Nguyen, H. T. (2015). Understanding abiotic stress tolerance mechanisms in soybean: A comparative evaluation of soybean response to drought and flooding

24

stress. Plant Physiology and Biochemistry, 86, 109–120. doi: 10.1016/j.plaphy.2014.11.010 Nakano, Y., & Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbatespecifc peroxidase in spinach chloroplasts. Plant and Cell Physiology, 22, 867– 880. doi: 10.1093/oxfordjournals.pcp.a076232 Ploschuk, R. A., Miralles, D. J., Colmer, T. D., Ploschuk, E. L., & Striker, G. G. (2018). Waterlogging of winter crops at early and late stages: impacts on leaf

ro of

physiology, growth and yield. Frontiers in Plant Science, 9, 1863. doi:10.3389/fpls.2018.01863

Posso, D. A., Borella, J., Reissig, G. N., & Bacarin, M. A. (2018). Root flooding-

-p

induced changes in the dynamic dissipation of the photosynthetic energy of

10.1007/s11738-018-2790-9

re

common bean plants. Acta Physiologiae Plantarum, 40, 212. doi:

lP

Ren, B., Zhang, J., Dong, S., Liu, P., & Zhao, B. (2016) Effects of Waterlogging on Leaf Mesophyll Cell Ultrastructure Summer Maize. PLoS ONE, 11, e0161424. doi: 10.1371/journal.pone.0161424

na

Rocha, M., Licausi, F., Araujo W. L., Nunes-Nesi, A., Sodek, L., Fernie, A. R., & van Dongen, J. T. (2010b). Glycolysis and the Tricarboxylic Acid Cycle Are

ur

Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of

Jo

Lotus japonicus. Plant Physiology, 152, 1501–1513. doi: 10.1104/pp.109.150045

Rocha, M., Sodek, L., Licausi, F., Hameed, M. W., Dornelas, M. C., & van Dongen, J. T. (2010a). Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilizers during hypoxic stress. Amino Acids, 39, 1043-1503. doi:10.1007/s00726-010-0596-1

25

Shimamura S., Mochizuki T., Nada Y., & Fukuyama M. (2002). Secondary aerenchyma formation and its relation to nitrogen fixation in root nodules of soybean plants (Glycine max) grown under flooded conditions. Plant Production Science, 5, 294–300. doi: 10.1626/pps.5.294 Shimamura, S., Yamamoto, R., Nakamura, T., Shimada, S., & Komatsu, S. (2010). Stem hypertrophic lenticels and secondary aerenchyma enable oxygen transport to roots of soybean in flooded soil. Annals of Botany, 106, 277–284. doi:

ro of

10.1093/aob/mcq123 Sousa, C. A. F., & Sodek, L. (2003). Alanine metabolism and alanine

aminotransferase activity in soybean (Glycine max) during hypoxia of the root

-p

system and subsequent return to normoxia. Environmental and Experimental Botany, 50, 1–8. doi: 10.1016/S0098-8472(02)00108-9

re

Tamang, B. G., Magliozzi, J. O., Maroof, M. A. S., & Fukao, T. (2014).

lP

Physiological and transcriptomic characterization of submergence and reoxygenation responses in soybean seedlings. Plant, Cell and Environment, 37, 2350–2365. doi: 10.1111/pce.12277

na

Thomas, A. L., Guerreiro, S. M. C., & Sodek, L. (2005). Aerenchyma formation and recovery from hypoxia of the flooded root system of nodulated soybean. Annals of

ur

Botany, 96, 1191–1198. doi: 10.1093/aob/mci272

Jo

United States Department of Agriculture. (2019). World Agricultural Production. Retrieved from https://apps.fas.usda.gov/psdonline/circulars/production.pdf

van Dongen, J. T., & Licausi, F. (2015). Oxygen sensing and signalling. Annual Review of Plant Biology, 66, 345–367. doi: 10.1146/annurev-arplant-043014114813

26

Van Nguyen, L., Takahashi, R., Githiri, S.M., Rodriguez, T.O., Tsutsumi, N., Kajihara, S., … Mochizuki, T. (2017). Mapping quantitative trait loci for root development under hypoxia conditions in soybean (Glycine max L. Merr.). Theoretical and Applied Genetics, 130, 743–755. doi: 10.1007/s00122-016-28473 Velikova, V., Yordanov, I., & Edreva, A. (2000). Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Science, 151, 59–66.

ro of

doi:10.1016/S0168-9452(99)00197-1 Vitor, S. C., & Sodek, L. (2019). Products of anaerobic metabolism in waterlogged roots of soybean are exported in the xylem. Plant Science, 284, 82–90. doi:

-p

10.1016/j.plantsci.2019.03.023

Wellburn, A. R. (1994). The spectral determination of chlorophylls a and b, as well

re

as total carotenoids, using various solvents with spectrophotometers of different

1617(11)81192-2

lP

resolution. Journal of Plant Physiology, 144, 307–313. doi: 10.1016/S0176-

Wollmer, A. C., Pitann, B., & Mühling, K. H. (2017). Waterlogging events during

na

stem elongation or flowering affect yield of oilseed rape (Brassica napus L.) but not seed quality. Journal of Agronomy and Crop Science, 204, 165–174. doi:

ur

10.1111/jac.12244

Jo

Xu, M., Ma, H., Zeng, L., Cheng, Y., Lu, G., Xu, J., …Zou, X. (2015). The effect of waterlogging on yield and seed quality at the early flowering stage in Brassica napus L. Field Crops Research, 180, 238–245. doi: 10.1016/j.fcr.2015.06.007

Ye, H., Song, L., Chen, H., Valliyodan, B., Cheng, P., Ali, L. ... Chen, P. (2018). A major natural genetic variation associated with root system architecture and

27

plasticity improves waterlogging tolerance and yield in soybean. Plant, cell & environment, 41, 2169–2182. doi: 10.1111/pce.13190 Young, A., & Lowe, G. (2018). Carotenoids-antioxidant properties. Antioxidants, 7, 28. doi: 10.3390/antiox7020028 Zabalza, A., van Dongen, J. T., Froehlich, A., Oliver, S. N., Faix, B., Gupta, K. J., … Geigenberger, P. (2009). Regulation of respiration and fermentation to control the plant internal oxygen concentration. Plant Physiology, 149, 1087–1098. doi:

ro of

10.1104/pp.108.129288 Zhang, P., Lyu, D., Jia, L., He, J., & Qin, S. (2017). Physiological and de novo

transcriptome analysis of the fermentation mechanism of Cerasus sachalinensis

-p

roots in response to short-term waterlogging. BMC genomics, 18, 649. doi:

Jo

ur

na

lP

re

10.1186/s12864-017-4055-1.

A

**

0.00

**

**

*

*

-0.05 -0.10 -0.15 -0.20 7W 7R

-0.25 -0.30

B

*

0.10

ro of

0.15

*

0.05

*

0.00

*

-0.05

-p

-0.10 -0.15 -0.20

re

0.20

C

*

*

*

*

lP

0.15

*

0.10 0.05 0.00 -0.05

6028

6042

6070

POT

ur

6000

na

SD [log10 (treatment/control)]

SDM [log10 (treatment/control)]

RDM [log10 (treatment/control)]

28

Genotype

Jo

FIGURE 1 Root dry matter (RDM) (A), shoot dry matter (SDM) (B) and stem diameter (SD) (C) for the different soybean genotypes at seven days of waterlogging (7W) and seven days of recovery (7R). Values are the ratio between treatment and control performed on log10-transformed data and represented the mean of five independent measurements. Asterisk (*) indicates a significant difference from the control (Student’s t-test; P ≤ 0.05).

* ***

0.0

** *

**

** *

**

-0.1 -0.2 -0.3

E [log10 (treatment/control)]

-0.4 0.2

C

*

*

0.1 0.0

*

***

**

**

-0.1 -0.2 -0.3 -0.4 6000

6028

6042

6070

B

*

0.2 0.0

**

** * *

*

*

-0.2 -0.4 -0.6 -0.8 0.2

D

*

0.1 0.0

**

-0.1 -0.2 -0.3 -0.4

6000

POT

***

**

*

6028

6042

6070

POT

-p

0.1

0.4

ro of

2W 7W 2R 7R

A

gs [log110 (treatment/control)]

0.2

WP [log10 (treatment/control)]

A [log10 (treatment/control)]

29

Genotype

re

Genotypes

lP

FIGURE 2 Net CO2 assimilation rate (A) (A), stomatal conductance (gs) (B), transpiration rate (E) (C) and water potential (WP) (D) at two (2W) and seven days

na

of waterlogging (7W), two (2R) and seven days of recovery (7R). Values are the ratio between treatment and control performed on log10-transformed data and

ur

represented the mean of five independent measurements. Asterisk (*) indicates a

Jo

significant difference from the control (Student’s t-test; P ≤ 0.05).

0.1

A

**

0.0

***

**

* *

*

-0.1 -0.2 2W 7W 2R 7R

-0.3 -0.4 B

0.0

**

*

ro of

0.1

*

-0.1 -0.2

-p

-0.3

0.2

*

0.1

* **

*

*

-0.1 -0.2 -0.3 -0.4

*

6028

6042

6070

POT

ur

6000

*

lP

0.0

re

-0.4

na

Carotenoids [log10 (treatment/control)]

Chl b [log10 (treatment/control)] Chl a [log10 (treatment/control)]

30

Genotypes

Jo

FIGURE 3 Level of chlorophyll a (Chl a) (A), chlorophyll b (Chl b) (B) and carotenoids (C) at two (2W) and seven days of waterlogging (7W), two (2R) and seven days of recovery (7R). Values are the ratio between treatment and control performed on log10-transformed data and represented the mean of five independent measurements. Asterisk (*) indicates a significant difference from the control (Student’s t-test; P ≤ 0.05).

Leaves 0.4

*

**

*

0.2

Col 6 Col 7 Col 8 * 9 Col

0.0

*

* *

* * *

0.0 2W 7W 2R 7R

-0.4

-0.2 -0.4

0.6 B

E

*

0.4

0.4

0.3

*

0.2

*

0.2

0.0

0.1

*

-0.2

0.0

-0.4

* **

re

*

-0.1

-0.6

-0.2

-0.8

*

* *

*

0.4

*

*

0.2

*

* *

na

-0.2

*

lP

C

0.2

-0.4 -0.6 -0.8 -1.0

*

-0.6

0.5

0.0

*

0.2

*

-0.2

0.4

D

**

ro of

A

Roots

-p

0.4

ur

CAT [log10 (treatment/control)]

APX [log10 (treatment/control)]

SOD [log10 (treatment/control)]

31

6000 6028 6042 6070 POT

*

*

0.0

*

-0.2

Col 6 Col 7 Col 8 Col 9

-0.4

* ** *

*

6028

6042

***

-0.6 -0.8 6000

6070

POT

Genotypes

Jo

Genotypes

F

FIGURE 4 Activity of superoxide dismutase (SOD) (A, D); ascorbate peroxidase (APX) (B, E); catalase (CAT) (C, F) in leaves (A, B, C) and roots (D, E, F) at two (2W) and seven days of waterlogging (7W), two (2R) and seven days of recovery (7R). Values are the ratio between treatment and control performed on log10transformed data and represented the means of five independent measurements.

32

Asterisk (*) indicates a significant difference from the control (Student’s t-test; P ≤ 0.05).

0.3

A

0.4

**

*

*

*

0.3

*

*

*

0.2

*

0.2

*

* 0.1

B

0.1

*

** *

0.0

0.0 2W 7W 2R 7R

-0.1 -0.2

*

-0.2

6000 6028 6042 6070 POT Genotypes

lP

Genotypes

-0.1

re

6000 6028 6042 6070 POT

*

ro of

0.4

Roots

-p

H2O2 [log10(treatment/control)]

Leaves

FIGURE 5 Level of hydrogen peroxide (H2O2) in leaves (A) and roots (B) at two

na

(2W) and seven days of waterlogging (7W), two (2R) and seven days of recovery (7R). Values are the ratio between treatment and control performed on log10-

ur

transformed data and represented the mean of four independent measurements. The

Jo

asterisk indicates a significant difference from the control (Student’s t-test; P ≤ 0.05).

* *

0.4 0.2

Col 6 Col 7 Col 8 Col 9

0.0 -0.2

*

* *

*

*

* 2W 7W 2R 7R

-0.4 -0.6 1.5 B

*

1.0

*

*

*

0.5

* *

*

*

0.0

*

-0.5 -1.0

6000

6028

6042

6070

POT

C

*

1.0

*

* *

0.5

* *

0.0 -0.5 -1.0 0.8

D

*

0.6 0.4 0.2 0.0 -0.2 -0.4

*

*

6000

6028

6042 6070

POT

Genotype

re

Genotype

1.5

ro of

0.6

A

-p

0.8

AlaAT [log10 (treatment/control)] ADH [log10 (treatment/control)]

PDC [log10 (treatment/control)] LDH [log10 (treatment/control)]

33

lP

FIGURE 6 Activity of lactate dehydrogenase (LDH) (A), pyruvate decarboxylase (PDC) (B), alcohol dehydrogenase (ADH) (C) and alanine aminotransferase (AlaAT)

na

(D) in roots at two (2W) and seven days of waterlogging (7W), two (2R) and seven days of recovery (7R). Values are the ratio between treatment and control performed

ur

on log10-transformed data and represented the mean of four independent measurements. The asterisk indicates a significant difference from the control

Jo

(Student’s t-test; P ≤ 0.05).